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SHELLFISH TOXICITY: HUMAN

HEALTH IMPLICATIONS OF

MARINE ALGAL TOXINS

Ms. Bébhine Carey

PROTEOBIO, Mass Spectrometry Centre, Cork


Institute of Technology and The Environmental
Research Institute, University College Cork.
OUTLINE
 Shellfish industry

 Harmful algal blooms (HAB‟s)

 Marine toxin syndromes


 Diarrhetic Shellfish Poisoning (DSP)

 Azaspiracid Shellfish Poisoning (AZP)

 Amnesic Shellfish Poisoning (ASP)

 Paralytic Shellfish Poisoning (PSP)

 Neurotoxic Shellfish Poisoning (NSP)

 Global increase in harmful algal blooms


SHELLFISH INDUSTRY
 Rich source

 protein, essential minerals, and

vitamins A and D

 Mussels, clams and scallops are

cultured at sea

 Bouchot culture method

 Raft culture method

 Bottom culture method

 Rack and bag culture


HARMFUL ALGAL BLOOMS (HAB‟S)
 Algae – usually autotrophic
organisms, unicellular or multi-
cellular
 Largest – seaweed

 Only organism to readily make


PUFAs

 Plankton – mass of micro-


organisms , drift or float
 Phytoplankton – diatoms,
dinoflagellates

 How a toxic algal bloom occurs:


HARMFUL ALGAL BLOOMS (HABS)
 Algae populations can increase rapidly
to form dense concentrations of cells
that may be visible.
 “Red Tides”

 Blooms are not always visible


 Discolouration varies with species of
phytoplankton, size and concentration,
and depth
 Phytoplankton generally proliferate
during the Summer months when the
water is calm and warmer.
HARMFUL ALGAL BLOOMS (HABS)
 Algal blooms can be non-toxic
providing energy to fuel food webs.
 Small percentage produce
powerful toxins
 Fish kills
 Death of mammals and birds
 Illness in humans

 HAB also include blooms of non-


toxic species
 Masses of algae die and decompose
 Depletion of oxygen content in water
 Oxygen levels become so low,
animals die or leave
MARINE TOXIN SYNDROMES
 Bivalve molluscs filter large volumes of water
 Mussels, clams, scallops
 Phytoplankton
 Toxic or non-toxic
 Accumulate in digestive gland
 Vectors

 Five major toxic syndromes:


 Diarrhetic Shellfish Poisoning (DSP)
 Azaspiracid Shellfish Poisoning (AZP)
 Amnesic Shellfish Poisoning (ASP)
 Paralytic Shellfish Poisoning (PSP)
 Neurotoxic Shellfish Poisoning (NSP)
Phytoplankton & Bacteria

Bivalves
Birds

Fish

Marine Mammals Human Crustaceans


MARINE TOXIN SYNDROMES
 First recorded fatal case:
 1793
 Captain George Vancouver
 Taboo to eat shellfish when the seawater became
phosphorescent due to dinoflagellate blooms
 Paralytic shellfish poisoning

 Lack of clinical testing methods


 Gross underestimation of the incidence of human
poisonings from algal toxins
 Many symptoms the same as bacterial or viral
infections
 Little known about chronic exposure to these
toxins
DIARRHETIC SHELLFISH POISONING (DSP)
 History:
 First documented incidence of Diarrhetic Shellfish
poisoning, occurred in 1976 in North-eastern Japan.
 164 people affected
 Diarrhoea, nausea, vomiting and abdominal pain
 Caused by ingestion of mussels and scallops
 The dinoflagellate, Dinophysis fortii was identified as the
source of the toxin
 Toxin responsible was named Dinophysistoxin (DTX)
 Legend in the region warned that mussels become toxic
during the month of the paulownia flowers (June)
DIARRHETIC SHELLFISH POISONING (DSP)
 Categorised into three structural
groups: O O
CH3 F
O H 31 G
Okadaic acid (OA) and
B C
 HO D O
O 35

OH
A O O O
dinophysistoxins (DTX) OH
E H
OH
HO
 Pectenotoxin (PTX)
 Yessotoxin (YTX)

 Other derivatives of okadaic acid:


 DTX 2 – an isomer of okadaic acid,
isolated from Irish mussels
 DTX 3 – isolated from the digestive
gland of the scallop Patinopecten
yessoensis
DIARRHETIC SHELLFISH POISONING (DSP)
 Toxins removed from DSP classification:
 Yessotoxins – sulphated polyether compounds, were isolated from
scallops along with DTX1 and DTX3

 Pectenotoxins – polyether macrolides, isolated from toxic scallops

 Do not cause diarrhoea

 Produce a very toxic response when injected i.p into mice

 No case of human poisoning has been reported


H 3C
CH3 O
H 3C HO
O O
O
O O
O
OH
Yessotoxin
OH O
OH CH3 O O
H3C O O CH3
O CH3
NaO3SO H 3C OH
O CH3 CH3
O O
O
O
Pectenotoxin 2 NaO3SO
O
O
CH3

O O
CH3
CH3
DIARRHETIC SHELLFISH POISONING (DSP)
 Symptoms:
 Diarrhoea, nausea, vomiting and abdominal cramps
 Onset of symptoms can occur within 30 mins up to a few
hours
 Complete recovery within 3 days
 Chronic effects – potent tumour promoters
 Toxicity studies – OA is two times more toxic than DTX2

 Often confused as bacterial enterotoxin poisoning


 Okadaic acid and dinophysistoxins are heat stable,
lipophilic compounds which accumulate in the
digestive glands of filter feeding bivalves
DIARRHETIC SHELLFISH POISONING (DSP)
 Produced by the dinoflagellates Dinophysis spp. and
Prorocentrum spp.
 Toxin profile varies within a single species

 Europe – OA and DTX2

 Japan – OA and DTX1

 Regulatory limit for these toxins in Europe is 0.16 µg/g

Prorocentrum Lima D. fortii D. acuminata D. acuta


AZASPIRACID SHELLFISH POISONING (AZP)
 Most recently discovered marine toxin

 Several analogues have been identified

 First confirmed incidence of Azaspiracid Shellfish poisoning,


occurred in 1995 in Netherlands.
 Caused by consumption of mussels (M. edulis)

 Cultivated in Killary Harbour, in the west of Ireland

 At least eight individuals were affected

 Abdominal pain, nausea, vomiting, diarrhoea – similar to DSP

 Azaspiracid 1 (AZA1) isolated from these mussels – its structure


identified
AZASPIRACID SHELLFISH POISONING (AZP)
 There are greater than 20 known analogues of azaspiracids
 Symptoms:
 Diarrhoea, nausea, vomiting and abdominal cramps
 Recovery within 3 days

 Toxicological studies
 Induce widespread organ damage in mice
 More dangerous than other classes of shellfish toxins
 Target organs – liver, spleen, the small intestine
 Carcinogenic
 Chronic exposure – tumour formation in the lungs and malignant
lymphomas
 Interstitial pneumonia
 Shortened small intestinal villi
AZASPIRACID SHELLFISH POISONING (AZP)
 Protoperidinium crassipes was initially thought to be the
causative agent
 Cannot produce their own food by photosynthesis

 Predators of dinoflagellates

 Do not proliferate into large blooms

 Vector of AZA toxins

 Azadium spinosum
 Dinoflagellate

 Identified as the producer of AZA toxins


AZASPIRACID SHELLFISH POISONING (AZP)
 AZA 2 recently found in a sponge
(Echinoclathria sp.) – Japan
 First report of AZA in Asia

 Global outbreaks:
 UK, Norway, France, Italy, Spain and Denmark
 North Africa

 Human intoxications due to AZP


 shellfish that had been passed as „safe for human
consumption‟ using DSP mouse bioassays.
 DSP mouse bioassay protocols result in the
extraction of only 5-40% of total azaspiracids present
in mussels.
AMNESIC SHELLFISH POISONING (ASP) CH3
 History:
COOH
 1987 in Canada H
COOH
 107 cases of shellfish poisoning COOH
CH3 N
 Vector responsible were mussels (M. edulis) H
 Cultivated on Prince Edward Island
 Victims suffered gastrointestinal disturbances as well as unusual
neurological symptoms – memory impairment
 3 people died within 18 days of admission
 Neurological symptoms – headaches, confusion, disorientation,
seizures and coma
 Permanent short term memory loss – Amnesic shellfish poisoning

 Domoic acid was identified as the toxin responsible


AMNESIC SHELLFISH POISONING (ASP)
 Epidemiological studies
 Age-dependant response
 Less than 40 years old – gastrointestinal problems
 Greater than 50 years old – suffer from memory
loss

 Onset of symptoms:
 Gastrointestinal – within 24hrs
 Neurological - within 48hrs
 Symptoms can last for a few days - possibility of
permanent memory loss

 Diatom of the Pseudonitzschia spp.


 In Japan, domoic acid previously known as a
natural product
 Anthelminthic and insecticidal properties
 Originally discovered in a seaweed
AMNESIC SHELLFISH POISONING (ASP)
 Marine animals
 1998 - 70 sea lions washed up onto beaches in California
 Suffering from neurological problems
 47 animals died
 DA identified in faecal samples and in nearby anchovies

 Birds
 1991 – DA poisoning report in Monterey Bay, California
 Pelicans and cormorants behaving strangely
 Vomiting, unusual head movements, scratching , many deaths

 Vector – northern anchovy


AMNESIC SHELLFISH POISONING (ASP)
 Alfred Hitchcock‟s “The Birds”.
 Similar event in 1961 in Santa Cruz,
prompted production
 Flocks of shearwaters – acting erratically,
flying into houses and cars, pecking
people, breaking windows, vomiting
 Reported in the local newspaper
 Clippings were included in Alfred Hitchcock's
studio proposal
 Based on a book by Daphne du Maurier

 Similar events have occurred since along


the same coastline
PARALYTIC SHELLFISH POISONING (PSP)
 PSP toxins are collectively called saxitoxins (STXs)
 At least 21 analogues of these cyclic guanidines

 Saxitoxin the most common

 Dinoflagellates responsible:
 Alexandrium, Gymnodinium, Pyrodinium species

H 2N O
H
O NH
HN NH

N NH
HN OH
OH
Alexandrium tamarense Structure of STX
PARALYTIC SHELLFISH POISONING (PSP)
 Symptoms:
 Mild – tingling sensation or numbness of the lips, face and neck.
Prickly sensation in fingertips and toes

 Severe – headache, nausea, vomiting, diarrhoea, muscular


paralysis and respiratory difficulty
 High risk of death in the absence of artificial respiration

 Onset of symptoms occurs rapidly

 Lethal dose is 1-4 mg STX or equivalents

 Saxitoxins clears from the blood within 24hrs

 No organ damage or long term effects

 Schedule 1 list of the Chemical Weapons Convention


PARALYTIC SHELLFISH POISONING (PSP)
 History:

 1927 – Northern California


 102 people poisoned from eating mussels

 6 deaths

 PSP outbreaks have occurred on both the eastern and


western coastlines of North America
 Alaska – badly affected

 PSP events for more than 130 years

 1987 – Cape Cod Bay


 14 humpback whales

 Mackerel
PARALYTIC SHELLFISH POISONING (PSP)
 Have been detected in European waters, human
intoxications are rare
 1970‟s – PSP intoxications from mussels cultivated in Spain,
Portugal and U.K
 80-120 individuals

 Repeated outbreaks in Chile and Argentina


 21 PSP deaths reported in Chile since 1991
 Rare identification of toxins in body fluids of victims

 Philippines
 2000 cases of PSP 1983-1998
 115 deaths
NEUROTOXIC SHELLFISH POISONING (NSP)
 Marine dinoflagellate – Karenia brevis
 Produce neurotoxins called brevetoxins
 Is a naked dinoflagellate – no protective layer
 Affect finfish, aquatic mammals and birds
 Death of large manatees and bottlenose dolphins

 Brevetoxin-producing HABs have caused problems in the Gulf of


Mexico for decades, records beginning in 1947
 NSP is not geographically widespread
HO
O
O
O
O
O O O
O
O O
O O

Brevetoxin structure Karenia brevis


NEUROTOXIC SHELLFISH POISONING (NSP)
 Symptoms:
 Act in three ways depending on route of
exposure – onset 30mins to 3hrs
 Symptoms persist for a few days
 Oral - gastroenteritis, chills, sweats,
reduction in core temperature, hypotension,
arrhythmias, numbness, peripheral tingling
and in severe cases broncho-constriction,
paralysis, seizures and coma.
 Inhalation – sea spray contains brevetoxins
due to delicate nature of Karenia brevis.
Causes irritation to the eyes and nasal
passages, respiratory problems
 Skin – eye and nasal irritation
NEUROTOXIC SHELLFISH POISONING (NSP)
 1987, the Gulf stream carried a Karenia
brevis bloom from Florida to North Carolina
 48 people with neurological symptoms

 Vector – oysters

 1 person was hospitalised with severe


neurological symptoms

 1993, New Zealand


 186 individuals affected with gastrointestinal
symptoms and respiratory problems

 Vectors – green mussels, cockles and oysters

 New brevetoxin analogues were detected


MOUSE BIOASSAY VS. ANALYTICAL TECHNIQUES
 Mouse Bioassay:

 Inject replicate mice with extract of sample

 Observe symptoms exhibited in mice

 Measure time it takes for the mice to die – overall toxicity

 Primitive, non-selective

 Results dependant on mouse strain, gender, age and weight –

results cannot be reproduced by other labs


MOUSE BIOASSAY VS. ANALYTICAL TECHNIQUES
100
PTX2
 Analytical techniques DTX2

Relative Abundance
 Can be validated

 Robust
OA
 Does not harm animals
00 1 2 3 4 5 6 7 8 9 10 11 12
 Sensitive and selective method Time (min)

 Regulatory limits:
 AZP and DSP – 0.16 µg/g

 ASP – 20 µg DA/g

 NSP – 0.8 µg brevetoxin/g

 PSP – 0.8 µg/g


GLOBAL INCREASE IN HAB‟S
 Positive effect:
 critical food for filter-feeding bivalve shellfish

 Negative effect:
 Major human health implications
 Environmental impact
 Severe economic losses to aquaculture, fisheries, and tourism.

 Factors affecting HABs growth are not well understood


 Explanations for possible increase:
 Increased scientific awareness
 Increased utilisation of coastal waters for aquaculture
 Eutrophication
 Climate change
 Increased global marine traffic
AZA

DSP
PSP
AZA AZA
Canada,
AZA
DSP
ASP PSP
PSP ASP Italy, 1997
ASP
DSP
DSP
PSP
ASP DSP
ASP
NSP PSP

ASP
AZA
PSP
ASP
PSP

ASP

DSP

PSP ASP
DSP PSP
DSP

NSP

DSP
PSP

DSP PSP ASP NSP AZP


INCREASED SCIENTIFIC AWARENESS
 Reports of HAB‟s, associated human illnesses, shellfish
closures – increased media attention
 More and more research is being carried out in the field
 Algae A. minutum only know to exist in Egypt until
1988
 Now it has been reported in Australia, Ireland, France,
Spain, Turkey, Portugal, Italy, east coast of North America,
Thailand, New Zealand, Taiwan and Japan

 DSP first documented in 1976 in Japan – Dinophysis


fortii
 In Europe - D. acuminata, D. acuta, D. norvegica etc.
 From 1976-1982, 1,300 DSP cases reported in Japan
 In 1981, 5,000 DSP cases reported in Spain
INCREASED SCIENTIFIC AWARENESS
 Research is driven with advances in analytical technology

 Mass spectrometry
 Single MS

 Triple quadrupole MS

 Ion Trap MS

 Time Of Flight MS

 Orbitrap technology
 Lower detection limits

 Much higher selectivity of compounds

 High mass accuracy

 Higher resolution than leading TOF


INCREASED COASTAL AQUACULTURE
 Overfishing of coastal waters – aquaculture
is the solution
 Increase in shellfish farming is resulting in
more reports of paralytic, diarrhetic,
neurotoxic and amnesic shellfish poisoning
 As a direct result of increased aquaculture
there is also an increase in biotoxin
monitoring
 Finfish culture – algae species which
damage gill tissue
 Increased aquaculture activities can lead to
localised nutrient enrichment
EUTROPHICATION
 Hong Kong harbour
 Eight fold increase in HAB‟s from
1976-1986
 Corresponded with increasing
human population and increase in
nutrient loading
 1972, Seto Inland Sea,
Chattonella antiqua bloom
 Fish farm
 Killed 14 million cultured yellow tail
fish
 Untreated sewage and industrial
waste from pulp and paper factories
 Introduced effluent controls to
reduce chemical oxygen demand
(COD)
 After a 4 year period:
 Frequency of blooms have decreased
EUTROPHICATION
 Changing land use – deforestation can also cause shifts in
phytoplankton species composition
 Increasing concentrations of humic substances in land run off

 Humic substances – end product of decayed matter

 Acid rain:
 Increase mobility of humic substances and trace metals in soil

 River water draining from agricultural land


 Rich in N and P

 Stimulates diatom growth

 River water draining from forest areas


 Rich in humic and fulvic acids

 Stimulate dinoflagellate blooms


EUTROPHICATION
 Nitrogen:phosphorus ratio – Tolo
harbour, Hong Kong, 1980‟s
 Molar ratio of N:P decreased nearly
50%
 Human population and sewage
loading increased
 Dinoflagellate blooms increased

 Altered N:P ratios implicated in


shifts in diatom-dominant to
dinoflagellate dominant blooms
 U.S , European and Asian coastlines
CLIMATE CHANGE
 Greenhouse effect and Warming of Oceans
 Dinoflagellate pyrodinium bahamense found
in coastal waters of the Atlantic and Indo-
West Pacific
 Survey of its resting cyst fossils - indicate
much wider distribution in the past
 In the Australasian region, dinoflagellate
does not extend further than Papua New
Guinea
 100,000 years ago it was found as far south as
Sydney harbour
 Concern that this toxic algae species will
return to Australian waters
CLIMATE CHANGE
 Extreme climate events
 NSP considered endemic to the Gulf of
Mexico and the east coast of Florida
 1987 major Florida outbreak
 Dispersed by the gulf stream northward into
North Carolina waters
 1993, 180 shellfish poisonings reported in
New Zealand similar to Karenia brevis
 Hidden plankton flora
 Developed into a bloom
 Triggered by unusual climatological
conditions
 El Nino event – imbalance atmospheric
pressure and sea temperature results in a
shoaling thermocline
CLIMATE CHANGE
 Extreme climate events
 hurricanes
 expand the existing distribution of cyst-producing toxic
dinoflagellates
 Alexandrium tamarense in New England after a 1972
hurricane

 Reef Disturbances
 Ciguatera finfish poisoning well-known in coral reef
areas in the Caribbean, Australia, and French Polynesia
 Rare disease two centuries ago
 Now it has reached epidemic proportions especially in
French Polynesia
 Reef disturbance by hurricanes, military, tourist
developments and coral bleaching (linked to global
warming) as well as future increasing coral damage due
to ocean acidification
 Increasing the risk of ciguatera.
INCREASED GLOBAL MARINE TRAFFIC
 Vector in dispersal of non-
indigenous marine plankton

 Release of ballast waters

 Algal cysts in ballast waters


 Planktonic stages of dinoflagellates -
limited survival during the voyage

 Resistant resting spores

 One single ballast tank – 300 million


toxic dinoflagellate cysts
INCREASED GLOBAL MARINE TRAFFIC
 PSP was unknown in Australia up until the
1980‟s
 First outbreak was in the ports of Hobart,
Melbourne and Adelaide

 Hobart – examination of historic plankton samples,


cyst surveys in dated sediment depth cores

 Toxic dinoflagellate G. Catenatum introduced after


1973

 Genetic fingerprinting using rRNA sequencing


 Genetic affinities between Australian and Japanese strains
of A. Catenella and Australian and European strains of A.
minutum

 Severe economic damage to fisheries


INCREASED GLOBAL MARINE TRAFFIC
 The International Maritime Organisation (IMO)
has introduced guidelines for ballast water
handling
 Reballasting at sea, ballasting in deep water ,
disposal of ballast tank sediment away from sensitive
aquaculture or marine park areas

 Ideally avoid ballasting during toxic dinoflagellate


blooms in ports

 Use heat, electrical shock or chemical treatment


INCREASED GLOBAL MARINE TRAFFIC
 Recent evidence
 first reports of palytoxin and tetrodotoxin in European waters
 Azaspiracids found in Japanese waters

 Tetrodotoxin
 Also called pufferfish poisoning
 Paralytic toxin
 Usually found in tropical and sub-tropical waters
 September 2007 - Trumpet shellfish (Charonia lampas sauliae) from
the southern coast of Portugal
 Symptoms include:
 perioral numbness, acral numbness, nausea, vomiting, dizziness or
vertigo, weakness, ataxia, dyspnea, diaphoresis, and death from
respiratory failure
INCREASED GLOBAL MARINE TRAFFIC
 Another possible vector:
 Translocation of shellfish stocks from one
area to another

 Faeces and digestive tracts of bivalves can


be loaded with:
 Viable dinoflagellates
 Resistant resting cysts

 Japanese seaweeds:
 Sargassum muticum (England, Netherlands,
Norway)
 Undaria pinnatifida and Laminaria
japonica (Mediterranean)
 Introduced to European waters through
Japanese oyster spat
CONCLUSION
 Impact on human health – increased in recent decades

 Increase of HAB‟s
 Increased marine traffic
 Climate change
 Eutrophication
 Aquaculture
 Increased scientific awareness

 Improvements in analytical techniques have limited toxin


exposure
 Clinical testing required
 James et al. Shellfish toxicity: human health implications of
marine algal toxins, epidemiology and infections, 2010

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